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Abstract:

The present disclosure relates to the use of nutritional compositions
including lactoferrin produced by a non-human source in stimulating
innate immune cells, such as macrophages, neutrophils, and dendritic
cells.

Claims:

1. A method for stimulating at least one type of innate immune cell in a
human comprising administering to the human a nutritional composition
comprising: a) a fat or lipid source; b) a protein source; and c)
lactoferrin produced by a non-human source, wherein the lactoferrin has
at least 48% homology with the amino acid sequence AVGEQELRKCNQWSGL at
the HLf (349-364) fragment, such that administration of the nutritional
composition stimulates at least one type of innate immune cell selected
from the group consisting of macrophages, neutrophils, dendritic cells,
and combinations thereof.

2. The method according to claim 1, wherein the nutritional composition
is administered to an infant or a child.

3. The method according to claim 1, wherein the fat or lipid source is
present at a level of about 3 g/100 kcal to about 7 g/100 kcal.

4. The method according to claim 1, wherein protein source is present at
a level of about 1 g/100 kcal to about 5 g/100 kcal.

5. The method according to claim 1, wherein the lactoferrin is present at
a level of at least about 10 mg/100 kcal.

6. The method according to claim 5, wherein the lactoferrin is present at
a level of about 70 mg/100 kcal to about 220 mg/100 kcal.

7. The method according to claim 1, wherein the lactoferrin is selected
from the group consisting of non-human lactoferrin, human lactoferrin
produced by a genetically modified organism, and combinations thereof.

8. The method according to claim 1, wherein the lactoferrin is stable and
remains active under conditions under which human lactoferrin becomes
unstable or inactive.

9. The method according to claim 8, wherein the nutritional composition
has been subject to pasteurization conditions.

10. The method according to claim 1, wherein the nutritional composition
further comprises a prebiotic composition comprising a compound selected
from the group consisting of galactooligosaccharide, polydextrose, and
combinations thereof.

11. The method according to claim 1, wherein the nutritional composition
comprises about 0.5 mg/100 kcal to about 5 mg/100 kcal of iron, including
iron bound to lactoferrin.

12. The method according to claim 1, wherein the nutritional composition
further comprises about 5 mg/100 kcal to about 100 mg/100 kcal of at
least one source of long chain polyunsaturated fatty acids.

13. The method according to claim 1, further comprising the step of
administering at least one probiotic.

Description:

BACKGROUND

[0001] 1. Technical Field

[0002] This disclosure relates generally to the field of nutritional
compositions, such as infant formulas, human milk fortifiers, children's
dietary supplements, and the like, having lactoferrin, in particular
lactoferrin produced by a non-human source. More particularly, the
disclosure relates to a method of stimulating innate immune cells in a
human by administering to the human nutritional compositions including
lactoferrin produced by a non-human source.

[0003] 2. Background

[0004] There are currently a variety of dietary compositions for humans,
especially young humans, to provide supplemental or primary nutrition at
certain stages in life. Generally, commercial dietary compositions for
infants seek to mimic to the extent possible the composition and
associated functionality of human milk. Through a combination of
proteins, some of which have physiological activity, and blended fat
ingredients, dietary compositions are formulated such that they simulate
human milk for use as a complete or partial substitute. Other ingredients
often utilized in dietary compositions for infants may include a
carbohydrate source such as lactose as well as other vitamins, minerals
and elements believed to be present in human milk for the absorption by
the infant.

[0005] Lactoferrin is one of the primary proteins in human milk and is
considered a glycoprotein having an average molecular weight of
approximately 80 kilodaltons. It is an iron binding protein having the
capacity to bind two molecules of iron in a reversible fashion and can
facilitate the uptake of iron within the intestines for the human.
Functionally, lactoferrin regulates iron absorption and as such can bind
iron-based free radicals as well as donate iron for an immunological
response.

[0006] An additional role of lactoferrin is its anti-microbial activity in
guarding against intestinal infections in humans generally, but
especially in infants. Lactoferrin has been known to be both
bacteriostatic and bactericidal in inhibiting the growth of specific
bacteria while also killing microbes prior to a successful invasion of
intestinal cells.

[0007] In obtaining a commercially viable dietary composition, the
addition of lactoferrin has generally been limited due to predicted
losses of activity during processing. For example, generally, the
temperature and pH requirements in processing infant formulas and other
products such as human milk fortifiers and various children's products
reduce specific functions of the lactoferrin, causing lactoferrin not to
be included within a final formulation. In addition, lactoferrin is often
considered only for its iron binding qualities; thus, lactoferrin may
generally be excluded from a formulation where such properties are
thought to be diminished by processing conditions.

[0008] Further, as known in the art, human breast milk is relatively low
in iron, containing about 0.3 milligrams of iron per liter of breast
milk. While this quantity is low, human infants have high absorption
rate, absorbing about half of the iron from the breast milk. However,
when human infants are given prior art formulas with high levels of iron
fortification, for example, of from about 10 mg to about 12 milligrams
per liter, the infants absorb less than about 5% of the total iron. With
such increased levels of iron within the prior art formulas, virtually
all of the iron binding sites would be expected to be occupied, as
lactoferrin is a known iron transport protein.

[0009] Additional complications of the prior art formulas include the
inability of providing a bacteriostatic effect. This is partially due to
the use of lactoferrin with blocked or damaged binding sites, as the
bacteriostatic effect is at least partially related to the degree of
binding to iron of the lactoferrin present within the formula.

[0010] Accordingly, it would be beneficial to provide a nutritional
composition, such as an infant formula, human milk fortifier, children's
dietary supplement, and the like, which contains lactoferrin, in
particular, lactoferrin produced by a non-human source. Preferably, the
lactoferrin included in the compositions is able to stimulate innate
immune cells even after processing under conditions of high temperature
and low pH. It also would be beneficial to determine the response of
innate immune cells to nutritional compositions including lactoferrin.

BRIEF SUMMARY

[0011] Briefly, the present disclosure is directed, in an embodiment, to a
method for stimulating at least one type of innate immune cell in a
human. In certain embodiments, the method comprises administering to the
human a nutritional composition comprising:

[0012] a. up to about 7 g/100 kcal of a fat or lipid source, more
preferably about 3 g/100 kcal to about 7 g/100 kcal of a fat or lipid
source;

[0013] b. up to about 5 g/100 kcal of a protein source, more preferably
about 1 g/100 kcal to about 5 g/100 kcal of a protein source; and

[0014] c. at least about 10 mg/100 kCal of lactoferrin, more preferably
about 70 mg to about 220 mg/100 kCal of lactoferrin, and most preferably
about 90 mg to about 190 mg/100 kCal of lactoferrin. Optionally, in
certain embodiments, the nutritional compositions may further comprise
about 0.1 g/100 kcal to about 1 g/100 kcal of a prebiotic composition
comprising polydextrose and/or galactooligosaccharide. More preferably,
the nutritional composition comprises about 0.3 g/100 kcal to about 0.7
g/100 kcal of a prebiotic composition which comprises a combination of
polydextrose and galactooligosaccharide.

[0015] Preferably, the lactoferrin is non-human lactoferrin and/or human
lactoferrin produced by a genetically modified organism. In one
particularly preferred embodiment, the lactoferrin used is such that an
effective amount of a nutritional composition containing lactoferrin may
be administered to stimulate at least one type of innate immune cell in a
human, even if, during processing, the nutritional composition has been
exposed to pH and temperature fluctuations typical of certain processing
conditions like pasteurization.

DETAILED DESCRIPTION

[0016] In certain embodiments, the present disclosure provides a method
for stimulating at least one type of innate immune cell in a human by
administering to the human nutritional compositions that comprise a lipid
or fat source, a protein source, and lactoferrin produced by a non-human
source.

[0017] As used herein, "lactoferrin from a non-human source" means
lactoferrin which is produced by or obtained from a source other than
human breast milk. For example, lactoferrin for use in the present
disclosure includes human lactoferrin produced by a genetically modified
organism as well as non-human lactoferrin. The term "organism", as used
herein, refers to any contiguous living system, such as animal, plant,
fungus or micro-organism. The term "non-human lactoferrin", as used
herein, refers to lactoferrin having an amino acid sequence that is
different than the amino acid sequence of human lactoferrin.

[0018] Lactoferrins are single chain polypeptides of about 80 kD
containing 1-4 glycans, depending on the species. The 3-D structures of
lactoferrin of different species are very similar, but not identical.
Each lactoferrin comprises two homologous lobes, called the N- and
C-lobes, referring to the N-terminal and C-terminal part of the molecule,
respectively. Each lobe further consists of two sub-lobes or domains,
which form a cleft where the ferric ion (Fe3+) is tightly bound in
synergistic cooperation with a (bi)carbonate anion. These domains are
called N1, N2, C1 and C2, respectively. The N-terminus of lactoferrin has
strong cationic peptide regions that are responsible for a number of
important binding characteristics. Lactoferrin has a very high
isoelectric point (˜pI 9) and its cationic nature plays a major
role in its ability to defend against bacterial, viral, and fungal
pathogens. There are several clusters of cationic amino acids residues
within the N-terminal region of lactoferrin mediating the biological
activities of lactoferrin against a wide range of microorganisms. For
instance, the N-terminal residues 1-47 of human lactoferrin (1-48 of
bovine lactoferrin) are critical to the iron-independent biological
activities of lactoferrin. In human lactoferrin, residues 2 to 5 (RRRR)
and 28 to 31 (RKVR) are arginine-rich cationic domains in the N-terminus
especially critical to the antimicrobial activities of lactoferrin. A
similar region in the N-terminus is found in bovine lactoferrin (residues
17 to 42; FKCRRWQWRMKKLGAPSITCVRRAFA).

[0019] As described in "Perspectives on Interactions Between Lactoferrin
and Bacteria" which appeared in the publication BIOCHEMISTRY AND CELL
BIOLOGY, pp 275-281 (2006), lactoferrins from different host species may
vary in their amino acid sequences though commonly possess a relatively
high isoelectric point with positively charged amino acids at the end
terminal region of the internal lobe. Suitable lactoferrins for use in
the present disclosure include those having at least 48% homology with
the amino acid sequence AVGEQELRKCNQWSGL at the HLf (349-364) fragment.
In some embodiments, the lactoferrin has at least 65% homology with the
amino acid sequence AVGEQELRKCNQWSGL at the HLf (349-364) fragment, and,
in embodiments, at least 75% homology. For example, non-human
lactoferrins acceptable for use in the present disclosure include,
without limitation, bovine lactoferrin, porcine lactoferrin, equine
lactoferrin, buffalo lactoferrin, goat lactoferrin, murine lactoferrin
and camel lactoferrin.

[0020] Lactoferrin for use in the present disclosure may be, for example,
isolated from the milk of a non-human animal or produced by a genetically
modified organism. For example, in U.S. Pat. No. 4,791,193, incorporated
by reference herein in its entirety, Okonogi et al. discloses a process
for producing bovine lactoferrin in high purity. Generally, the process
as disclosed includes three steps. Raw milk material is first contacted
with a weakly acidic cationic exchanger to absorb lactoferrin followed by
the second step where washing takes place to remove nonabsorbed
substances. A desorbing step follows where lactoferrin is removed to
produce purified bovine lactoferrin. Other methods may include steps as
described in U.S. Pat. Nos. 7,368,141, 5,849,885, 5,919,913 and
5,861,491, the disclosures of which are all incorporated by reference in
their entirety.

[0021] In one embodiment, lactoferrin is present in the nutritional
composition in an amount of at least about 10 mg/100 kCal, especially
when the nutritional composition is intended for use by children. In
certain embodiments, the upper limit for lactoferrin is about 240 mg/100
kCal. In another embodiment, where the nutritional composition is an
infant formula, lactoferrin is present in the nutritional composition in
an amount of from about 70 mg to about 220 mg/100 kCal; in yet another
embodiment, lactoferrin is present in an amount of about 90 mg to about
190 mg/100 kCal. Nutritional compositions for infants can include
lactoferrin in the quantities of from about 0.5 mg to about 1.5 mg per
milliliter of formula. In nutritional compositions replacing human milk,
lactoferrin may be present in quantities of from about 0.6 mg to about
1.3 mg per milliliter of formula.

[0022] As mentioned, in certain embodiments of the disclosure, the
nutritional compositions stimulate at least one type of innate immune
cell when administered to humans. Several types of innate immune cells
are well-known in the art and include, without, limitation, natural
killer cells, mast cells, eosinophils, basophils; and the phagocytic
cells including macrophages, neutrophils and dendritic cells. Preferably,
when administered to humans, the nutritional compositions stimulate
macrophages, dendritic cells and/or neutrophils. The nutritional
compositions may stimulate the innate immune cells by any means known in
the art. Preferably, the nutritional compositions stimulate the
expression of costimulatory molecules from macrophages and/or dendritic
cells. In another preferred embodiment, the nutritional compositions
stimulate the production of cytokines from macrophages and/or dendtritic
cells. In yet another preferred embodiment, the nutritional compositions
stimulate the respiratory burst capacity of neutrophils.

[0023] Preferably, the lactoferrin used in the nutritional composition
retains its stability and activity in the human gut even after processing
under conditions of high temperature and low pH. In one embodiment of the
present disclosure, the lactoferrin used is non-human lactoferrin.

[0024] For example, surprisingly, bovine lactoferrin maintains certain
bactericidal activity even if exposed to a low pH (i.e., below 7, and
even as low as about 4.6 or lower) and/or high temperatures (i.e., above
about 65° C., and as high as about 120° C.), conditions
which would be expected to destroy or severely limit the stability or
activity of human lactoferrin. These low pH and/or high temperature
conditions can be expected during certain processing regimen for
nutritional compositions of the types described herein, such as
pasteurization. Yet, while bovine lactoferrin has an amino acid
composition which has about a 70% sequence homology to that of human
lactoferrin, and is stable and remains active under conditions under
which human lactoferrin becomes unstable or inactive, bovine lactoferrin
has bactericidal activity against undesirable bacterial pathogens found
in the human gut.

[0025] In some embodiments, the nutritional compositions of the disclosure
may be an infant formula. The term "infant formula" applies to a
composition in liquid or powdered form that satisfies the nutrient
requirements of an infant by being a substitute for human milk. In the
United States, the content of an infant formula is dictated by the
federal regulations set forth at 21 C.F.R. §§100, 106 and 107.
These regulations define macronutrient, vitamin, mineral, and other
ingredient levels in an effort to simulate the nutritional and other
properties of human breast milk. In a separate embodiment, the
nutritional product may be a human milk fortifier, meaning it is a
composition which is added to human milk in order to enhance the
nutritional value of human milk. As a human milk fortifier, the disclosed
composition may be in powder or liquid form. In yet another embodiment,
the disclosed nutritional product may be a children's nutritional
composition.

[0026] Preferably, the nutritional composition is administered to an
infant or a child. As used herein, the term "infant" is generally defined
as a human from birth to 12 months of age. A "child" and "children" are
defined as humans over the age of 12 months to about 12 years old.

[0027] The nutritional compositions of the disclosure may provide minimal,
partial, or total nutritional support. The nutritional compositions may
be nutritional supplements or meal replacements. In some embodiments, the
nutritional compositions may be administered in conjunction with a food
or another nutritional composition. In this embodiment, the nutritional
compositions can either be intermixed with the food or other nutritional
composition prior to ingestion by the subject or can be administered to
the subject either before or after ingestion of a food or nutritional
composition. The nutritional compositions may be administered to preterm
infants receiving infant formula, breast milk, a human milk fortifier, or
combinations thereof. For purposes of the present disclosure, a "preterm
infant" is an infant born after less than 37 weeks gestation, while a
"full term infant" is an infant born after at least 37 weeks gestation.

[0028] The nutritional compositions may, but need not, be nutritionally
complete. The skilled artisan will recognize "nutritionally complete" to
vary depending on a number of factors including, but not limited to, age,
clinical condition, and dietary intake of the subject to whom the term is
being applied. In general, "nutritionally complete" means that the
nutritional composition of the present disclosure provides adequate
amounts of all carbohydrates, lipids, essential fatty acids, proteins,
essential amino acids, conditionally essential amino acids, vitamins,
minerals, and energy required for normal growth. As applied to nutrients,
the term "essential" refers to any nutrient which cannot be synthesized
by the body in amounts sufficient for normal growth and to maintain
health and which therefore must be supplied by the diet. The term
"conditionally essential" as applied to nutrients means that the nutrient
must be supplied by the diet under conditions when adequate amounts of
the precursor compound is unavailable to the body for endogenous
synthesis to occur.

[0029] The composition which is "nutritionally complete" for the preterm
infant will, by definition, provide qualitatively and quantitatively
adequate amounts of all carbohydrates, lipids, essential fatty acids,
proteins, essential amino acids, conditionally essential amino acids,
vitamins, minerals, and energy required for growth of the preterm infant.
The composition which is "nutritionally complete" for the full term
infant will, by definition, provide qualitatively and quantitatively
adequate amounts of all carbohydrates, lipids, essential fatty acids,
proteins, essential amino acids, conditionally essential amino acids,
vitamins, minerals, and energy required for growth of the full term
infant. The composition which is "nutritionally complete" for a child
will, by definition, provide qualitatively and quantitatively adequate
amounts of all carbohydrates, lipids, essential fatty acids, proteins,
essential amino acids, conditionally essential amino acids, vitamins,
minerals, and energy required for growth of a child.

[0030] The nutritional composition may be provided in any form known in
the art, including a powder, a gel, a suspension, a paste, a solid, a
liquid, a liquid concentrate, or a ready-to-use product. In one preferred
embodiment, the nutritional composition is an infant formula, especially
an infant formula adapted for use as sole source nutrition for an infant.

[0031] In the preferred embodiments, the nutritional product disclosed
herein may be administered enterally. As used herein, "enteral" means
through or within the gastrointestinal, or digestive, tract, and "enteral
administration" includes oral feeding, intragastric feeding, transpyloric
administration, or any other introduction into the digestive tract.

[0034] In one embodiment, the proteins are provided as intact proteins. In
other embodiments, the proteins are provided as a combination of both
intact proteins and partially hydrolyzed proteins, with a degree of
hydrolysis of between about 4% and 10%. In yet another embodiment, the
protein source may be supplemented with glutamine-containing peptides.

[0035] In a particular embodiment of the disclosure, the protein source
comprises whey and casein proteins and the ratio of whey to casein
proteins ratio is similar to that found in human breast milk. For
example, in certain embodiments, the weight ratio of whey to casein
proteins is from about 20% whey:80% casein to about 80% whey:20% casein.

[0036] In one embodiment of the disclosure, the nutritional composition
may contain one or more probiotics. The term "probiotic" means a
microorganism with low or no pathogenicity that exerts beneficial effects
on the health of the host. Any probiotic known in the art may be
acceptable in this embodiment provided it achieves the intended result.
In a particular embodiment, the probiotic may be selected from
Lactobacillus species, Lactobacillus rhamnosus GG, Bifidobacterium
species, Bifidobacterium brevis, Bifidobacterium longum, and
Bifidobacterium animalis subsp. lactis BB-12.

[0037] If included in the composition, the amount of the probiotic may
vary from about 104 to about 1010 colony forming units (cfu)
per kg body weight per day. In another embodiment, the amount of the
probiotic may vary from about 106 to about 109 cfu per kg body
weight per day. In yet another embodiment, the amount of the probiotic
may be at least about 106 cfu per kg body weight per day. Moreover,
the disclosed composition may also include probiotic-conditioned media
components.

[0038] In one embodiment, one or more of the probiotics is viable. In
another embodiment, one or more of the probiotics is non-viable. As used
herein, the term "viable" refers to live microorganisms. The term
"non-viable" or "non-viable probiotic" means non-living probiotic
microorganisms, their cellular components and metabolites thereof. Such
non-viable probiotics may have been heat-killed or otherwise inactivated
but retain the ability to favorably influence the health of the host. The
probiotics useful in the present disclosure may be naturally-occurring,
synthetic or developed through the genetic manipulation of organisms,
whether such new source is now known or later developed.

[0039] In one embodiment of the disclosure, the nutritional compositions
may include a prebiotic composition comprising one or more prebiotics. As
used herein, the term "prebiotic" means a non-digestible food ingredient
that beneficially affects the host by selectively stimulating the growth
and/or activity of one or a limited number of bacteria in the colon that
can improve the health of the host. A "prebiotic composition" is a
composition that comprises one or more prebiotics. Such prebiotics may be
naturally-occurring, synthetic, or developed through the genetic
manipulation of organisms and/or plants, whether such new source is now
known or developed later. In certain embodiments, the prebiotic included
in the compositions of the present disclosure include those taught by
U.S. Pat. No. 7,572,474, the disclosure of which is incorporated herein
by reference.

[0041] If included in the nutritional compositions, the total amount of
prebiotics present in the nutritional composition may be from about 0.1
g/100 kcal to about 1 g/100 kcal. More preferably, the total amount of
prebiotics present in the nutritional composition may be from about 0.3
g/100 kcal to about 0.7 g/100 kcal. At least 20% of the prebiotics should
comprise galactooligosaccharide (GOS) and/or polydextrose (PDX).

[0042] If polydextrose is used in the prebiotic composition, the amount of
polydextrose in the nutritional composition may, in an embodiment, be
within the range of from about 0.1 g/100 kcal to about 1 g/100 kcal. In
another embodiment, the amount of polydextrose in the nutritional
composition is within the range of from about 0.2 g/100 kcal to about 0.6
g/100 kcal.

[0043] If galactooligosaccharide is used in the prebiotic composition, the
amount of galactooligosaccharide in the nutritional composition may, in
an embodiment, be from about 0.1 g/100 kcal to about 1 g/100 kcal. In
another embodiment, the amount of galactooligosaccharide in the
nutritional composition is from about 0.2 g/100 kcal to about 0.5 g/100
kcal. In certain embodiments, the ratio of polydextrose to
galactooligosaccharide in the prebiotic composition is between about 9:1
and about 1:9.

[0044] The nutritional formulation of the disclosure, in some embodiments,
may further contain a source of long chain polyunsaturated fatty acids
(LCPUFAs). Preferably, the source of LCPUFAs comprise docosahexanoic acid
(DHA). Other suitable LCPUFAs include, but are not limited to,
α-linoleic acid, γ-linoleic acid, linoleic acid, linolenic
acid, eicosapentanoic acid (EPA) and arachidonic acid (ARA).

[0045] In one embodiment, the nutritional composition is supplemented with
both DHA and ARA. In this embodiment, the weight ratio of ARA:DHA may be
from about 1:3 to about 9:1. In one embodiment of the present disclosure,
the weight ratio of ARA:DHA is from about 1:2 to about 4:1.

[0046] The amount of long chain polyunsaturated fatty acids in the
nutritional composition may vary from about 5 mg/100 kcal to about 100
mg/100 kcal, more preferably from about 10 mg/100 kcal to about 50 mg/100
kcal.

[0047] The nutritional composition may be supplemented with oils
containing DHA and ARA using standard techniques known in the art. For
example, DHA and ARA may be added to the composition by replacing an
equivalent amount of an oil, such as high oleic sunflower oil, normally
present in the composition. As another example, the oils containing DHA
and ARA may be added to the composition by replacing an equivalent amount
of the rest of the overall fat blend normally present in the composition
without DHA and ARA.

[0048] If utilized, the source of DHA and ARA may be any source known in
the art such as marine oil, fish oil, single cell oil, egg yolk lipid,
and brain lipid. In some embodiments, the DHA and ARA are sourced from
the single cell Martek oil, DHASCO® and ARASCO® respectively, or
variations thereof. The DHA and ARA can be in natural form, provided that
the remainder of the LCPUFA source does not result in any substantial
deleterious effect on the infant. Alternatively, the DHA and ARA can be
used in refined form.

[0049] In an embodiment of the present disclosure, sources of DHA and ARA
are single cell oils as taught in U.S. Pat. Nos. 5,374,567; 5,550,156;
and 5,397,591, the disclosures of which are incorporated herein in their
entirety by reference. However, the present disclosure is not limited to
only such oils.

[0050] In certain embodiments, the nutritional compositions comprise from
about 0.5 mg/100 kcal to about 5 mg/100 kcal of iron, including iron
bound to lactoferrin.

EXAMPLES

[0051] The following examples are provided to illustrate embodiments of
the nutritional composition of the present disclosure but should not be
interpreted as any limitation thereon. Other embodiments within the scope
of the claims herein will be apparent to one skilled in the art from the
consideration of the specification or practice of the nutritional
composition or methods disclosed herein. It is intended that the
specification, together with the example, be considered to be exemplary
only, with the scope and spirit of the disclosure being indicated by the
claims which follow the examples.

Example 1

[0052] This example illustrates an embodiment of a nutritional product
according to the present disclosure.

[0056] Preferably, the nutritional composition is administered to a human
and stimulates at least one type of innate immune cell in the human.

[0057] All references cited in this specification, including without
limitation, all papers, publications, patents, patent applications,
presentations, texts, reports, manuscripts, brochures, books, internet
postings, journal articles, periodicals, and the like, are hereby
incorporated by reference into this specification in their entireties.
The discussion of the references herein is intended merely to summarize
the assertions made by their authors and no admission is made that any
reference constitutes prior art. Applicants reserve the right to
challenge the accuracy and pertinence of the cited references.

[0058] Although preferred embodiments of the disclosure have been
described using specific terms, devices, and methods, such description is
for illustrative purposes only. The words used are words of description
rather than of limitation. It is to be understood that changes and
variations may be made by those of ordinary skill in the art without
departing from the spirit or the scope of the present disclosure, which
is set forth in the following claims. In addition, it should be
understood that aspects of the various embodiments may be interchanged
both in whole or in part. For example, while methods for the production
of a commercially sterile liquid nutritional supplement made according to
those methods have been exemplified, other uses are contemplated.
Therefore, the spirit and scope of the appended claims should not be
limited to the description of the preferred versions contained therein.

Patent applications by Anja Wittke, Evansville, IN US

Patent applications in class Amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same

Patent applications in all subclasses Amino acid sequence disclosed in whole or in part; or conjugate, complex, or fusion protein or fusion polypeptide including the same